Pravastatin is an HMG-CoA reductase inhibitor. Pravastatin sodium has been used in the treatment of hyperlipemia or hyperlipidaemia and has the useful pharmacological effect of being able to reduce serum cholesterol. Pravastatin can be obtained using Streptomyces carbophilus by microbial conversion of ML-236B produced by Penicillium citrinum [described in Endo, A., et al., J. Antibiot., 29 1346(1976): Matsuoka, T., et al., Eur. J. Biochem., 184, 707 (1989), and in Japanese Patent Application Publication No 57-2240].
It has been shown that both ML-236B, a precursor of pravastatin, and lovastatin, a HMG-CoA inhibitor, share the same partial structure. They are synthesized biologicals via polyketides [described in Moore, R. N., at al., J. Am. Chem. Soc., 107, 3694(1985); Shiao, M, and Don, H. S., Proc. Natl. Sci. Counc. Repub. China B. 11, 223(1987)].
Polyketides are compounds derived from a β-keto carbon chains that result from a continuous condensation reaction of low-molecular weight carboxylic acids, such as acetic acid, propionic acid, butyric acid or the like. Various structures may be derived depending on the pathway of condensation or reduction of each of the β-keto carbonyl groups [described in Hopwood, D. A, and Sherman, D. H., Annu. Rev. Genet., 24, 37–66 (1990): Hutchinson, C. R. and Fujii, I., Annu. Re. Microbiol., 49, 201–238(1995)].
Polyketide Synthases (hereinafter referred to as PKSs) that contribute to the synthesis of polyketides are enzymes known to be present in filamentous fungi and bacteria. The enzymes of filamentous fungi have been studied using molecular biological techniques [as described in Feng, G. H, and Leonard, T. J., J. Bacteriol., 177, 6246 (1995); Takano, Y., et al. Mol. Gen. Genet. 249, 162 (1995)]. In Aspergillus terreus, which is a lovastatin producing micro-organism, a PKS gene related to the biosynthesis of lovastatin has been analyzed [described in International application laid-open in Japan (KOHYO) No.9-504436, and see corresponding WO 9512661 which claims DNA encoding a triol polyketide synthase].
Genes related to biosynthesis of secondary metabolites of filamentous fungi often form a cluster on the genome. In the pathways of the biosynthesis of polyketides, gene clusters participating in said pathway are known to exist. In the biosynthesis of Aflatoxin, which is a polyketide produced by Aspergillus flavus and Aspergillus parasiticus, genes encoding enzyme proteins participating in said biosynthesis (such as PKS) have been known to form a cluster structure. Genomic analysis and a comparison of the genes participating in the biosynthesis of Aflatoxin in each of the micro-organisms has been carried out [see Yu, J., et al., Appl. Environ. Microbiol., 61, 2365 (1995)]. It has been reported that genes participating in biosynthesis of Sterigmatocystin produced by Aspergillus nidulans form a cluster structure in about 60 kb of a continuous region on its genome [described in Brown, D. W, et al., Proc. Natl. Acad. Sci. USA, 93, 1418 (1996)].
The modulation of polyketide synthase activity by accessory proteins during lovastatin synthesis has been investigated [see Kennedy, J, et al. Science Vol 284, 1368 (1999)].
However, to date, there has been insufficient molecular biological analysis into the biosynthesis of ML-236B, and factors regulating it. The present invention sets out to address this problem